JP2002507397A - HLA binding peptides and uses thereof - Google Patents

Abstract

  (57) [Summary] The present invention relates to an immunogenic peptide, which specifically binds to the glycoprotein encoded by the HLA allele and is capable of inducing T cell activation in T cells restricted by that allele. Means and methods of selection and the immunogenic peptide composition are provided. The present peptides are useful for eliciting an immune response against a desired antigen.

Description

DETAILED DESCRIPTION OF THE INVENTION

BACKGROUND OF THE INVENTION The present invention relates to compositions and methods for the prevention, treatment or diagnosis of a number of pathological conditions, such as viral diseases and cancer. In particular, the present invention provides novel peptides that can bind to selected major histocompatibility complex (MHC) molecules and elicit an immune response.

[0002] MHC molecules are classified as either class I or class II molecules. Class II MHC molecules are mainly expressed on cells involved in initiating and sustaining an immune response, such as T lymphocytes, B lymphocytes, macrophages, and the like. Class II MHC molecules are recognized by helper T cells and induce the proliferation of helper T lymphocytes and the amplification of the immune response to the particular immunogenic peptide presented. Class I MHC molecules are expressed on almost all nucleated cells and are recognized by cytotoxic T lymphocytes (CTLs), which are then destroyed by antigen-bearing cells. CTLs are particularly important in tumor rejection and in fighting viral infections.

[0003] CTLs recognize antigens in the form of peptide fragments bound to MHC class I molecules more than the intact foreign antigen itself. Antigens usually must be synthesized endogenously by cells, and some of the protein antigens are broken down into small peptide fragments in the cytoplasm. Some of these small peptides are pre-Golgi (pre-G
olgi) transported into compartments and interacts with class I heavy chains to facilitate proper folding and association with subunit β2 microglobulin. This peptide-MHC class I complex is then transported to the cell surface for expression and potential recognition by specific CTLs.

Investigation of the crystal structure of the human MHC class I molecule, HLA-A2.1, indicates that a peptide-binding groove is created by folding the α1 and α2 domains of the class I heavy chain. (Bjorkman et al., Nature 329: 5
06 (1987)). However, in these studies, the identity of the peptide binding to the groove was not determined.

[0005] Buus et al., Science 242: 1065 (1988) first described a method for acid elution of bound peptides from MHC. Later, Rammensee and co-workers (Falk et al
., Nature 351: 290 (1991)) developed an approach to characterize naturally processed peptides bound to class I molecules. Other investigators have reported that the B-type (Jardetzky, et al., Nature 353: 326 (1991)) and the A2.1-type by mass spectrometry (Hunt, et al., Science 225: 1261 (1992)). )) By means of conventional automated sequencing of peptides eluted from class I molecules.
Direct amino acid sequencing of the larger amount of peptide within the PLC fraction has been successfully achieved. A review of the characterization of naturally processed peptides in MHC class I can be found in Roetzschke and Falk (Roetzschke and Falk, Immunol. Today 12: 447
(1991)).

Natl. Acad. Sci. USA 86: 3296 (1989) Sette et al., MHC allele-specific motif could be used to predict MHC binding capacity. It was shown that. Scieffer et al., Proc. Natl. Acad. Sc . USA 86: 4649 (1989) have shown that MHC binding is involved in immunogenicity. Some authors (De Bruijn et al., Eur. J. Immunol ., 21: 2963-2970
(1991); Pamer et al., 991 Nature 353: 852-955 (1991)) provide a preliminary report that class I binding motifs can be applied to the identification of potentially immunogenic peptides in animal models. Provided proof. Class I motifs specific to multiple human alleles of a given class I isotype have not yet been described. Desirably, the frequency of these different allele combinations must be high enough to cover a large fraction or perhaps the majority of the human outbred population.

[0007] Despite the developments in the field, the prior art has not yet provided useful human peptide-based vaccines or therapeutics based on the above studies. The present invention provides these and other advantages. SUMMARY OF THE INVENTION The present invention provides a composition comprising an immunogenic peptide having a binding motif for an HLA molecule. Immunogenic peptides that bind to the appropriate MHC allele are
Conserved residues are included at specific positions that allow the peptide to bind to the desired HLA molecule.

[0008] Epitopes on a number of immunogenic target proteins can be identified using the peptides of the present invention. Examples of suitable antigens include prostate cancer specific antigen (PSA)
), Hepatitis B core and surface antigens (HBVc, HBVs), hepatitis C antigen, Eps
tein-Barr virus antigen, human immunodeficiency type 1 virus (HIV1), Kaposi's sarcoma herpes virus (KSHV), human papilloma virus (HP
V) Antigen, Lassa virus, Mycobacterium tuberculosis (M. tuberculosis MT), p53, CEA, trypanosomal surface antigen (TSA)
), And Her2 / neu. Thus, the peptides are useful in pharmaceutical compositions for both therapeutic and diagnostic applications.

[0009] In particular, the present invention relates to the present invention, wherein the immunogenic peptide is a peptide shown in Tables 3 to 14,
Compositions comprising an immunogenic peptide having an HLA binding motif are provided. Table 3-
Also provided are peptides containing conservative substitutions of residues within the peptide set forth in 14. The immunogenic peptide of the present invention can be further linked to a second oligopeptide. In some aspects, the second oligopeptide is a peptide that includes a helper T response.

[0010] The present invention further provides nucleic acids encoding immunogenic peptides as set forth in Tables 3-14, or peptides comprising conservative substitutions of the residues of the peptides set forth in Tables 3-14. The nucleic acid can further include a sequence encoding a second immunogenic peptide or a peptide that elicits a helper T response. The peptides provided herein can be used to elicit a cytotoxic T cell response either in vivo or in vitro. The method comprises contacting cytotoxic T cells with a peptide of the invention.

Definitions The term "peptide" refers to a series of residues, typically L-amino acids, typically joined together by a peptide bond between the alpha-amino and carbonyl groups of adjacent amino acids. For that reason, it is used interchangeably herein with “oligopeptide”. The oligopeptides of the present invention are less than about 15 residues in length, and usually consist of about 8 to about 11 residues, preferably 9 or 10 residues.

“Immunogenic peptide” refers to a peptide that binds to an MHC molecule, and
Peptides containing an allele-specific motif so as to elicit a response. The immunogenic peptides of the present invention can bind to the appropriate HLA molecule and elicit a cytotoxic T cell response to the antigen from which the immunogenic peptide is derived.

[0013] Immunogenic peptides are conveniently identified using the algorithm of the present invention. This algorithm provides a grade (score) that allows the selection of immunogenic peptides
Is a mathematical procedure. Typically, one skilled in the art will have a "binding threshold" that allows for the selection of peptides that have a high probability of binding at a particular affinity and will then be immunogenic. Use the above algorithm class. The algorithm is based on either the effect of a particular amino acid at a particular position on the peptide on MHC binding, or on the binding of a particular substitution within a motif-containing peptide.

“Conserved (residual) residues” are amino acids that occur at a significantly higher frequency which will be predicted by a random distribution at a particular position in the peptide. Typically, the conserved residues are such that their MHC structure can provide the immunogenic peptide at the point of contact. At least 1 to 3 in a peptide of a predetermined length
Thus, preferably two, conserved residues define a motif for the immunogenic peptide. These residues are typically in close contact with the peptide binding groove, and their side chains are buried in specific pockets of the groove. Typically, an immunogenic peptide will contain up to three conserved residues, more usually two conserved residues.

As used herein, “negative binding residues”
"Is a non-bonded or weak binder if present at a particular location
Binders) and are subsequently immunogenic, ie, amino acids that will fail to elicit a CTL response. The term "motif" refers to the pattern of residues in a peptide of a given length, usually having from about 8 to about 11 amino acids, recognized by a particular MHC allele. This peptide motif typically differs for each human MHC allele, and differs in the pattern of highly conserved and negative residues.

[0016] The binding motif for the allele has a high degree of increasing degrees of precision.
ision). In some cases, all of the conserved residues are at the correct position in the peptide and are located, with no negative residues at 3 and / or 7. The phrase "isolated" or "biologically pure" refers to a material that is substantially or essentially free of components that normally accompany it, as found in its natural state. Thus, the peptides of the present invention are free of materials normally associated with their in situ environment, such as MHC I molecules on antigen presenting cells. Protein
Even when isolated to a homogeneous or predominant band, trace contaminants are present in the range of 5-10% of the native protein, which is co-purified with the desired protein. The isolated peptides of the present invention do not include such endogenous co-purified proteins.

The term “residue” refers to an amino acid or amino acid mimic incorporated into an oligopeptide by an amide bond or amide bond mimic. DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention relates to the determination of allele-specific peptide motifs for human class I MHC allele subtypes (sometimes referred to as HLA), particularly peptide motifs recognized by HLA alleles.

For HLA-A2.1, a 9 amino acid peptide preferably has the following motif: at the second position from the N-terminus selected from the group consisting of I, V, A, and T A first conserved amino residue and a second conserved residue at the C-terminal position selected from the group consisting of V, L, I, A, and M. Another motif is that the first conserved residue at the second position from the selected N-terminus is from the group consisting of L, M, I, V, A, and T, and the selected C-terminus The second conserved residue at the position is from the group consisting of A and M. The amino acid at position 1 is preferably D
And P are not selected from the group consisting of The third amino acid from the N-terminus is not an amino acid selected from the group consisting of D, E, R, K, and H.
The sixth amino acid from the N-terminus is not an amino acid selected from the group consisting of R, K, and H. The seventh amino acid from the N-terminus is R, K, H, D, and E
Not an amino acid selected from the group consisting of:

The HLA-A2.1 binding motif for a peptide having 10 residues is as follows: 2 from the N-terminus selected from the group consisting of L, M, I, V, A, and T A second conserved residue at the C-terminal position selected from the group consisting of V, I, L, A, and M; The first and second conserved residues are 7 residues apart. Preferably, the amino acid at position 1 is not an amino acid selected from the group consisting of D, E, and P. The N-terminal residue is not an amino acid selected from the group consisting of D and E. Residues at position 4 from the N-terminus are A, K, R, and H
Not an amino acid selected from the group consisting of: The amino acid at position 5 from the N-terminus is not P. The amino acid at position 7 from the N-terminus is not an amino acid selected from the group consisting of R, K, and H. The amino acids at position 8 from the N-terminus are D, E, R
, K, and H are not selected from the group consisting of: The amino acid at position 9 from the N-terminus is not an amino acid selected from the group consisting of R, K, and H.

The motif for HLA-A3.2 is the first conserved residue from L, M, I, V, S, A, T, and F at position 2 from its N-terminus to the C-terminus. Group and its C-
Contains a second conserved residue from K, R, or Y at the end. The other first conserved residues are C, G
Or D, and or E. Other second conserved residues are H or F. The first and second conserved residues are preferably 6-7 residues apart.

The motif for HLA-A1 comprises, from its N-terminus to the C-terminus, T, S,
Or a first conserved residue of M, a second conserved residue of D or E, and a third conserved residue of Y. Other second conserved residues are A, S or T. The first conserved residue and the second conserved residue are contiguous and preferably are separated from the third conserved residue by as much as 6-7 residues. Second
The motif consists of a first conserved residue from E or D and a second conserved residue from Y, wherein the first and second conserved residues are 5 to 6 residues apart.

The motif for HLA-A11 is from position T, V, M, L, I, S, A, G, N, C, D or F at position 2 from its N-terminus to the C-terminus. As well as the C-terminal conserved residue from K, R, Y or H. The first and second conserved residues are preferably 6 or 7 residues apart. The motif for HLA-A24.1 consists of two motifs from its N-terminus to the C-terminus.
Position, the first conserved residue from Y, F or W, and the C from F, I, W, M or L
-Contains terminal conserved residues. The first and second conserved residues are preferably 6 to 7
Residues apart.

In turn, these motifs may be associated with T cell epitopes from any desired antigen, especially human viral diseases, cancer or autoimmune diseases, for which potential antigens or Used to define what the amino acid sequence of the self antigen target is known. A number of potential target protein epitopes can be identified in this manner. Examples of suitable antigens include prostate specific antigen (PSA), hepatitis B core and surface antigens (HBVc, HBVs), hepatitis C antigen, Epstein-Barr virus antigen, melanoma antigen (eg MAGE-1), human Immunodeficiency virus (HIV) antigen, human pepiroma virus (HPV) antigen, Lassa (Las)
sa) Virus, Mycobacterium tuberculosis (M. tuberculosis MT)
, P53, CEA, trypanosomal surface antigen (TSA), and Her2 / neu
including.

[0024] Peptides containing epitopes from the antigens are synthesized and then, for example, by immunofluorescent staining and flow microfluorometry, peptide-dependent class I assembly assays, and inhibition of CTL recognition by peptide competition. For example, their ability to bind to appropriate MHC molecules is tested in assays using cells expressing pure class I molecules and radioiodinated peptides, and / or empty class I molecules. A peptide that binds to the class I molecule,
For their ability to serve as targets for CTLs obtained from infected or immunized individuals, as well as to generate CTL populations that can react with virtually infected target cells or tumor cells as potential therapeutics. Their ability to elicit a primary in vitro or in vivo CTL response that can be assessed is evaluated.

The MHC class I antigen is encoded by the HLA-A, B, and C loci.
HLA-A and B antigens are expressed on the cell surface at approximately equal densities, while
LA-C expression is significantly lower (probably 10-fold lower). Each of the above loci has multiple alleles. The peptide binding motifs of the present invention are relatively specific for each allele subtype.

For peptide-based vaccines, the peptides of the present invention preferably contain a motif recognized by MHC I molecules that are widely distributed in the human population. Since this MHC allele occurs at different frequencies in different races (ethnic groups and races), the choice of target MHC allele can depend on the target population. Table 1 shows the frequency of each allele in the HLA-A locus product between different races. For example, the majority of Caucasian populations can be covered by peptides that bind to four HLA-A allele subtypes, especially HLA-A2.1, A1, A3.2, and A24.1. Similarly, the majority of the Asian population is encompassed by the addition of peptides that bind to the fifth allele HLA-A11.2.

[0027]

[Table 1]

The nomenclature used to describe the peptide compounds follows the following convention. Here, the amino group is represented to the left (N-terminus) and the carboxyl group is represented to the right (C-terminus) of each amino acid residue. In the formulas representing selected specific embodiments of the present invention, unless otherwise indicated, the amino- and carboxyl end groups are in the form they will exhibit at physiological pH unless otherwise stated. In the amino acid structural formulas, each residue is generally represented by standard three letter or one letter nomenclature. The L-form of amino acid residues is represented by a capital letter or the first capital letter of the three-letter symbol, and the D-form of those amino acids having the D-form is represented by a single lowercase letter or a lowercase three-letter symbol. . Glycine has no asymmetric carbon atom and is simply "
Gly "or G.

The procedure used to identify the peptides of the invention is generally described by Falk et al.
., Nature 351: 290 (1991), which is incorporated herein by reference. Briefly, these methods involve MH from appropriate cells or cell lines, typically by immunoprecipitation or affinity chromatography.
Includes large-scale isolation of C class I molecules. Alternative examples for the isolation of desired MHC molecules, equally well known to those skilled in the art, include ion exchange chromatography, lectin chromatography, size exclusion, high performance ligand chromatography, and all combinations of the above techniques. Including.

[0030] Typically, immunoprecipitation is used to isolate the desired allele. Many protocols can be used, depending on the specificity of the antibody used. For example, allele-specific mAb reagents are HLA-A, HLA-B ₁ ,
And HLA-C molecules for affinity purification. H
Several mAb reagents are available for the isolation of LA-A molecules. Monoclonal BB7.2 is suitable for isolating the HLA-A2 molecule. Affinity columns prepared with the mAbs using standard techniques have been successfully used to purify the corresponding HLA-A allele product.

In addition to allele-specific mAbs, anti-HLA-A, B,
C mAbs, e.g., anti-HLA-B of W6 / 32 and B9.12.1, and 1
, C mAb, B1.23.2 could be used in other affinity purification protocols as described in the earlier application. Peptides bound to the peptide binding groove of the isolated MHC molecule are typically eluted using acid treatment. Peptides can also be dissociated from Class I molecules by a variety of standard denaturing means, such as heat, pH, detergents, salts, chaotropic agents, or combinations thereof.

[0032] The peptide fraction is further separated from MHC molecules by reverse phase high performance liquid chromatography (HPLC) and sequenced. Peptides can be produced by various other methods well known to those skilled in the art, including filtration, ultrafiltration, electrophoresis, size chromatography, precipitation with specific antibodies, ion exchange chromatography, isoelectric focusing, and others. It can be separated by standard means.

[0033] Sequencing of the isolated peptides is accomplished by standard techniques, such as Edman degradation (H
unkapiller, M.W.,et al.,Methods Enzymol. 91, 399 [1983])
Can be Other suitable methods for sequencing are as described above.
And mass spectrometry sequencing of individual peptides (Hunt, et al., Science 225: 1261 (1992), which is incorporated herein by reference). Different classes
Amino acid of bulk foreign peptide (eg pooled HPLC fraction) from molecule I
Noic acid sequencing typically involves the identification of sequence motifs characteristic of each class I allele.
Represent.

The definition of a motif specific for different class I alleles allows the identification of potential peptide epitopes from antigenic proteins whose amino acid sequence is known. Typically, identification of potential peptide epitopes is first performed using a computer to scan the amino acid sequence of the desired antigen for the presence of the motif. Next, an epitope sequence is synthesized. The ability to bind to MHC class molecules is measured in a variety of different ways. One means is a class I molecule binding assay as described in the related application above. An alternative method described in the above references is to inhibit antigen presentation (Sette, et al., J. Immunol . 141: 3893 (19
91)), in vitro assembly assay (Townsend, et al., Cell 62:28).
5 (1990)), and mutated cells, such as RMA. Includes a FACS-based assay using S (Melief, et al., Eur. J. Immunol. 21: 2963 (1991)).

Next, peptides that test positive in the MHC class I binding assay are assayed for their ability to induce a specific CTL response in vitro. For example, antigen presenting cells incubated with the peptide can be assayed for its ability to induce a CTL response in the responder cell population. Antigen presenting cells can be normal cells, for example, peripheral blood mononuclear cells or dendritic cells (Inaba, et al., J. Exp.Med. 166: 182 (1987); Boog, Eur. Im munol. 18: 219 [1988]).

Alternatively, a mutant mammalian cell line, such as the mouse cell line RMA, which is defective in their ability to load class I molecules with internally processed peptides
-S (Kaerre, et al., Nature , 319: 675 (1986); Ljunggren, et al., Eur. J. Immunol. 21: 2963-2970 (1991)), and human somatic T cell hybrids,
T-2 (Cerundolo, et al., Nature 345: 449-452 (1990)), as well as mutant mammalian cell lines transfected with the appropriate human class I gene, to which peptides are added. Sometimes it is conveniently used to test for the ability of the peptide to elicit a primary CTL response in vitro. Other eukaryotic cell lines that could be used include various insect cell lines, mosquito larvae (
ATCC cell line CCL 125, 126, 1660, 1591, 6585, 65
86), silkworm (ATCC CRL 8851), and armyworm (ATCC CR)
L 1711), moths (ATCC CCL 80), and Drosophila (Dros)
ophila) cell line, for example, the Schneider cell line (see Schneider J. Embryol. Exp. Mor phol . 27: 353-365 [1927]).

Peripheral blood lymphocytes are conveniently isolated following simple venipuncture or leukocyte separation of normal donors or patients and used as a source of responder cells for CTL precursors. In one embodiment, the appropriate antigen-presenting cells are incubated with 10-100 μM peptide in serum-free medium for 4 hours under appropriate culture conditions. The peptide-loaded antigen presenting cells are then incubated with the responder cell population in vitro for 7-10 days under optimized culture conditions. Positive CTL activation is detected by radiolabeled target cells, ie, specific peptide-pulsed targets,
As well as assaying the culture for the presence of CTLs that kill both the target virus expressing the endogenously processed form of the relevant virus or tumor antigen from which the peptide sequence is derived. .

[0038] The specificity of CTLs and MHC restriction are determined by testing against different peptide target cells expressing appropriate or inappropriate human MHC class I. Peptides that yield a positive result in the MHC binding assay and produce a specific CTL response are referred to herein as immunogenic peptides. The immunogenic peptide can be prepared synthetically or by recombinant DNA technology or from a natural source, such as a whole virus or tumor. The peptide will preferably be substantially free of other native host cell proteins and fragments thereof, but in some embodiments, the peptide can be synthetically linked to a native fragment or particle. .

The polypeptides or peptides can have varying lengths, either in their neutral (uncharged) or salt form, and can be modified, glycosylated, side chain oxidized, or They can either be unphosphorylated or include these modifications, provided that the modifications do not destroy the biological activity of the polypeptide as described herein.

Desirably, the peptide will be as small as possible while still retaining substantially all of the biological activity of the large peptide. Where possible, the peptides of the invention are 9 or 10 amino acid residues in length, in size to match endogenously processed viral or tumor cell peptides that bind to MHC class I molecules on the cell surface. It is desirable to optimize

Certain desirable properties, such as improvement, while increasing or at least retaining substantially all of the biological activity of the unmodified peptide that binds to the desired MHC molecule and activates appropriate T cells If it is necessary to provide the desired pharmacological properties, the peptide with the desired activity can be modified. For example, the peptides may have various alterations, whether conservative or non-conservative, where such alterations may provide certain advantages in their use, such as improved MHC binding. , For example, can be substituted. A conservative substitution is the substitution of one amino acid residue for another that is biologically and / or chemically similar, eg, 1
Substitution of one hydrophobic residue for another, or one polar residue for another. The above substitution is performed by Gly, Ala; Val, Ile, Leu, Met; Asp, G
lu; Asn, Gln; Ser, Thr; Lys, Arg; and Phe, Tyr.
Including combinations of The effect of a single amino acid substitution can also be probed using D-amino acids. Such modifications are described, for example, in Merrifield, Science 232:
341-347 (1986), Barany and Merrifield, The Peptides , Gross and Meienhof
er, eds. (NY, Academic Press), pp. 1-284 (1979); and Stewart and You
ng, Solid Phase Peptide Synthesis, (Rockford, Ill., Pierce), 2d Ed.
4) can be carried out using well-known peptide synthesis procedures, such as those described in US Pat.

The above peptides can also be modified by extending or shortening the amino acid sequence of the compound, for example, by adding or deleting amino acids. The peptides or analogs of the present invention can also be modified by changing the order or composition of particular residues. Certain amino acid residues essential for biological activity, such as those at critical contact sites or conserved residues, generally cannot be changed without adversely affecting biological activity That is easy to understand. Non-critical amino acids are those that are naturally found in the protein, for example, L-α
-It is not necessary to be limited to amino acids or their D-isomers, but can also include unnatural amino acids, for example β-γ-δ-amino acids as well as many derivatives of L-α-amino acids.

Typically, a series of peptides with single amino acid substitutions are used to determine the effect of electrostatic, hydrophobicity on binding. For example, a series of positively charged (eg, Lys or Arg) or negatively charged (eg, Glu) amino acid substitutions are made along the length of a peptide that exhibit different patterns of sensitivity to various MHC and T cell receptors. . Furthermore, small, comparative neutral components, such as A
Multiple substitutions using la, Gly, Pro, or similar residues can be used. These substitutions can be homo-oligomers or hetero-oligomers. The number and type of residues substituted or added will depend on the essential points of contact and the spatial arrangement required between the specific functional properties sought (eg, hydrophobic versus hydrophilic). High binding affinity for MHC molecules or T cell receptors also
It can be achieved by such substitutions as compared to the affinity of the parent peptide. In each case, such substitutions should employ, for example, amino acid residues or other molecular fragments that are chosen to avoid steric and electrical interference, which might break the bond.

[0044] Amino acid substitutions typically have a single residue. Substitutions, deletions, insertions, or any combination thereof, can be combined to achieve the final peptide. Substitution variants are such that at least one residue of the peptide has been removed and a different residue inserted in its place. Such substitutions are generally made according to Table 2, below, when it is desired to fine tune the properties of the peptide.

[0045]

[Table 2]

Function (eg, affinity for MHC molecules or T cell receptors)
The substantive change in is that by selecting a substitution that is less conservative than that in Table 2, ie, (a) the structure of the peptide backbone within the region of the substitution, such as, for example, a sheet or helical configuration , (B) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain, by choosing residues that differ more significantly in their effect on maintaining. In general, substitutions that are expected to produce the greatest change in peptide properties are that (a) a hydrophilic residue, such as seryl, is replaced by a hydrophobic residue, such as leucyl, isoleucyl, phenylalanyl, valyl, or alanyl. (B) a residue having a positively charged side chain, such as lysyl, arginyl or histidyl, substituted by a negatively charged residue; such as glutamyl or aspartyl; or (c) bulky A residue having a side chain, eg, phenylalanine, would be replaced by a residue without a side chain, eg, glycine.

The peptide may comprise two or more residue isosteres (iso) within the immunogenic peptide.
(steres). As defined herein, isostar
A-is a sequence of two or more residues that can be replaced with a second sequence. why
If the configuration of the first sequence fits the binding site specific to the second sequence
It is. The term "specifically" refers to peptide backbone modifications well known to those skilled in the art.
including. Such modifications include amide nitrogen, α-carbon, amide carbonyl,
Includes complete substitution, extension, deletion or backbone bridge modification of the bond. In general, Spatola, Chemistry and Biochemistry of Amino Acids, peptides and Proteins , Vol. V
II (Weinstein ed., 1983).

Modification of a peptide with various amino acid mimics or unnatural amino acids is particularly useful in increasing the stability of the peptide in vivo. Stability can be assayed in a number of ways. For example, peptidases and various biological media, such as human plasma and serum, have been used to test stability. For example, Verhoef et al., Eur. J. Drug Metab. Pharmacokin, 11: 291.
See -302 (1986). The half-life of the peptides of the present invention is conveniently measured using a 25% human serum (v / v) assay. This protocol is generally as follows. Pooled human serum (type AB, non-heat inactivated) is defatted by centrifugation before use. This serum is then diluted to 25% with RPMI tissue culture medium and used to test peptide stability. At predetermined time intervals, a small amount of the reaction solution is removed and added to either 6% aqueous trichloroacetic acid or ethanol. The cloudy reaction sample is cooled (4 ° C.) for 15 minutes, and then spun to pellet the precipitated serum proteins. The presence of the peptide is then determined by reverse-phase H using stability-specific chromatography conditions.
Measure by PLC.

The peptides of the invention or analogs thereof having CTL stimulating activity can be modified to provide desired properties other than improved serum half-life. For example, C
The ability of the peptide to elicit TL activity can be enhanced by ligation to a sequence containing at least one epitope capable of eliciting a T helper cell response. Particularly preferred immunogenic peptide / T helper conjugates (conjugates)
) Are linked by a spacer molecule. The spacer is typically a relatively small, neutral molecule that is substantially unaltered under physiological conditions, e.g.,
Consists of amino acids or amino acid mimics. These spacers are typically selected, for example, from Ala, Gly, or other neutral spacers of non-polar or neutral polar amino acids. It will be appreciated that the optional spacers need not consist of the same residues, and can therefore be hetero- or homo-oligomers. When present, the spacer will usually be at least one or two residues, more usually three to six residues. Alternatively, the CTL
The peptide can be linked to the T helper peptide without a spacer.

The immunogenic peptide can be linked to a T helper peptide either directly at the amino or carboxy terminus of the CTL peptide or via a spacer. The amino terminus of either the immunogenic peptide or the T helper peptide can be acylated. Exemplary T helper peptides are tetanus toxin 830-843, influenza 307-
319, circumsporozoite
382-398 and 378-389.

In some embodiments, it is desirable to include at least one component that primes CTL in the pharmaceutical compositions of the present invention. Lipids have been identified as agents that can prime CTL in vivo against viral antigens. For example,
The palmitic acid residue can be linked to the alpha and epsilon amino groups of the Lys residue, and then, for example, to one or more linking residues, eg, Gl
It can be linked to an immunogenic peptide via y, Gly-Gly, Ser, Ser-Ser, and others. The lipidated peptide can then be injected directly in micellar form, incorporated into liposomes, or emulsified in an adjuvant, such as incomplete Freund's adjuvant. In a preferred embodiment, a particularly effective immunogen is via a linkage, eg, Ser-Ser,
Lys alpha attached to the amino terminus of the immunogenic peptide and palmitic acid attached to the epsilon amino group.

As another example of lipid stimulation of the CTL response, Coli (E. coli) lipoprotein, e.g. tripalmitoyl -S- glyceryl Resid cysteinyl glyceryl - serine (P ₃ CS
S) can be covalently linked to a suitable peptide and used to stimulate virus-specific CTL. See Deres et al., Nature 342: 561-564 (1989).
This is incorporated herein by reference). The peptide of the present invention can specifically stimulate a CTL response to a target antigen by, for example, binding to P ₃ CSS and administering to a subject as a lipopeptide. In addition, P ₃ CSS conjugated to a peptide that presents the appropriate epitope can also stimulate the induction of neutralizing antibodies, so that the combination of the two compositions allows for both humoral and cellular responses to infection. Can be caused more efficiently.

In addition, additional amino acids may be added to the termini of the peptide in order to facilitate the coupling of the peptides to one another, to couple to a carrier support or large peptide, to alter the physical or chemical properties of the peptide or oligopeptide, etc. Can be added. Amino acids such as tyrosine, cysteine, lysine, glutamic acid or aspartic acid and the like can be introduced at the C-terminus or N-terminus of the peptide or oligopeptide. Modifications at the C-terminus may optionally alter the binding properties of the peptide. Additionally, the peptide or oligopeptide sequences terminated NH ₂ acylation, e.g., alkanoyl (C ₁ -C ₂₀₎ or thio glycolyl acetylation, terminal carboxyl amidation, e.g., ammonia, by modifying the methylamine and the like, natural The sequence can be different. In some cases, those modifications may provide a site for attachment to a support or another molecule.

[0054] The peptides of the present invention can be prepared in various ways. Due to their relatively short size, it is possible to synthesize peptides in solution or on a solid support according to conventional techniques. Various automatic synthesizers are commercially available and can be used according to known protocols. See, e.g., Stewart & Young, Solid Phase Peptide Synthesis , Second Edition, Pierce Chemical Co. (1984) supra.

Alternatively, recombinant DNA technology may be used. In that case, the nucleotide sequence encoding the immunogenic peptide of interest is inserted into an expression vector, suitable host cells are transformed or transfected with the expression vector, and the cells are transformed under conditions suitable for expression. Incubate with Such techniques are generally well known in the art and are described in Sambrook et al . , Molecular Cloning. A Laboratory Manual , Co.
ld Spring Harbor Press, Cold Spring Harbor, New York (1982), which is incorporated herein by reference. Thus, the present invention can use a fusion protein comprising one or more peptide sequences of the present invention to present an appropriate T cell epitope.

The coding sequence for a peptide of the expected length in the present invention can be synthesized by chemical techniques, for example, by the phosphotriester method of Matteucci et al . , J. Am. Chem. Soc . 103: 3185 (1981). Therefore, the modification can be easily performed by replacing one or more bases encoding the native peptide sequence with appropriate bases. The desired fusion protein is then provided by providing a suitable linker to the coding sequence, ligating into an expression vector commercially available in the art, and transforming the appropriate host with the vector. Can be produced. Many such vectors and suitable host systems are currently available. For expression of the fusion protein, an operably linked start and stop codon, promoter and terminator regions, and usually a replication system, are provided for the coding sequence to provide an expression vector for expression in the desired cellular host. Will be done. For example, a promoter sequence compatible with the bacterial host may be provided in a plasmid containing convenient restriction sites for insertion of the desired coding sequence. The resulting expression vector is used to transform a suitable bacterial host. Of course, yeast and mammalian cell hosts can also be used by using appropriate vectors and regulatory sequences.

The peptides of the present invention and pharmaceutical and vaccine compositions thereof are useful for administration to mammals, especially humans, for treating and / or preventing viral infection and cancer. Examples of diseases that can be treated using the immunogenic peptides of the present invention include prostate cancer, hepatitis B, hepatitis C, AIDS, kidney cancer, cervical cancer, lymphoma, CMV, condyloma acuminatum).

In the case of a pharmaceutical composition, the immunogenic peptide of the invention is administered to an individual already infected with the virus of interest or having cancer. Individuals in the latent or acute phase of the infection, if appropriate, can be treated with the immunogenic peptide individually or in combination with another treatment. For therapeutic use, the compositions are administered to a patient in an amount sufficient to elicit an effective CTL response to a viral or tumor antigen and to cure or at least alleviate the symptoms and / or complications. An amount sufficient to accomplish this is defined as "therapeutically effective amount." The effective amount for this use is
It will usually depend on the peptide composition, the mode of administration, the stage and severity of the disease to be treated, the weight and overall health of the patient, and the judgment of the attending physician, but will usually be about 1% for a 70 kg patient. An initial dose of peptide from 0.0 μg to about 5000 μg (ie, therapeutic or prophylactic administration) followed by measurement of specific CTL activity in the patient's blood can range from weeks to months depending on the patient's response and circumstances. A booster dose of about 1.0 μg to about 1000 μg of peptide is used according to the booster treatment. It must be kept in mind that the peptides and compositions of the invention can generally be used in severe disease states, ie, life-threatening or potentially life-threatening conditions. In such cases, it is possible and also desirable to administer a substantial excess of those peptide compositions in that the peptides are relatively non-toxic and the amount of foreign material is minimal. The physician may feel.

For therapeutic use, administration should begin at the first sign of viral infection or upon detection or surgical resection of the tumor or immediately after diagnosis in the case of acute infection. This is followed by a booster treatment at least until the symptoms subside substantially and further over a period of time thereafter. In the case of a chronic infection, a booster dose may be required following the loading dose.

Treatment of an infected individual with a composition of the invention can hasten resolution of the infection in acutely infected individuals. For individuals susceptible to (or susceptible to) chronic infection, the compositions of the present invention are particularly useful in methods of preventing progression from acute to chronic infection. Where a susceptible individual is identified before or during infection, for example, as described herein, the compositions of the invention can be specifically targeted to such individuals, and The need to administer to a population can be minimized.

The peptide compositions can be used for the treatment of chronic infections and for stimulating the immune system to remove virus infected cells in virus carriers. It is important to provide a sufficient amount of immunopotentiating peptide in the formulation and an administration method to efficiently stimulate a cytotoxic T cell response. Thus, for the treatment of chronic infections, a typical dose is from about 1.0 μg to about 5000 μg for a 70 kg patient at a time.
g, preferably in the range of about 5 to 1000 μg.

Subsequent to the immunization dose, booster doses at fixed intervals, eg, every 1 to 4 weeks, may be necessary for a longer period of time, possibly to effectively immunize the individual. In the case of chronic infection, administration should be continued at least until clinical symptoms or laboratory tests indicate that the viral infection has been eliminated or substantially resolved, and for a period of time thereafter.

Pharmaceutical compositions for therapeutic treatment are prepared for parenteral, topical, oral or local administration. Preferably, the pharmaceutical compositions are administered parenterally, for example, intravenously, subcutaneously, intradermally or intramuscularly. Thus, the present invention provides a composition for parenteral administration comprising a solution of the immunogenic peptide dissolved or suspended in a suitable carrier, preferably an aqueous carrier. Various aqueous carriers such as water, buffered water, 0.8% saline, 0.3% glycine,
Hyaluronic acid or the like can be used. These compositions may be sterilized by conventional, well-known sterilization techniques, or may be sterile filtered. The resulting aqueous solutions can be packaged for use as is, or lyophilized, the lyophilized preparation being mixed with a sterile solution prior to administration. The compositions may contain pharmaceutically acceptable auxiliary substances necessary to approximate physiological conditions, such as pH adjusters and buffers, toxicity adjusters, wetting agents, such as sodium acetate, sodium lactate, sodium chloride, potassium chloride, It may contain calcium chloride, sorbitan monolaurate, triethanolamine, triethanolamine oleate, and the like.

The concentration of the CTL stimulating peptide of the present invention in the pharmaceutical composition can vary widely,
That is, less than about 0.1% by weight, usually at least or about 2% by weight to 20-
It may range up to as much as 50% by weight, and will be selected according to the particular mode of administration chosen, for example mainly by volume, viscosity and the like. The peptides of the present invention can be administered via liposomes. Liposomes serve not only to direct the peptide to specific tissues, such as lymphoid tissues, or specifically to infected cells, but also to increase the half-life of the peptide composition. Liposomes include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, multilayers, and the like. The peptide to be delivered may be alone or with a molecule that binds to it, for example, a monoclonal antibody that binds to a receptor widely present in lymphoid cells, such as the CD45 antigen.
It is incorporated into those formulations as part of the liposome. The liposomes used in the present invention are composed of standard vesicle-forming lipids, usually comprising neutral phospholipids and negatively charged phospholipids and sterols such as cholesterol.
The choice of lipids is made, for example, by considering the size of the liposome, acid instability and the stability of the liposome in the bloodstream. Various methods can be used to prepare liposomes, for example, Szoka et al . , Ann. Rev. Biophys. Bioeng. 9: 467 (1980); U.S. Pat. Nos. 4,235,871, 4,501,728, and 4; , 837,028 and 5,019,369, which are incorporated herein by reference.

The ligand to be incorporated into the liposome to target immune cells includes, for example, an antibody specific to a cell surface determinant of a desired immune system cell or a fragment thereof. The liposome suspension containing the peptide is administered intravenously, topically, topically, etc., in different dosages depending, inter alia, on the mode of administration, the peptide to be delivered and the stage of the disease to be treated.

Solid compositions include, for example, medicinal mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose,
Conventional non-toxic solid carriers such as sucrose, magnesium carbonate and the like can be used. For oral administration, commonly used excipients, for example, any of the carriers enumerated above, and usually from 10 to 95% of the active ingredient, ie, one or more peptides of the invention, preferably from 25% to 75% A pharmaceutically acceptable non-toxic composition is prepared by including a concentration of the peptide of the invention.

For aerosol administration, the immunogenic peptides of the invention are provided in finely divided form, preferably together with a surfactant and propellant. A typical peptide ratio is 0.01
% By weight, preferably 1% by weight to 10% by weight. Of course, the surfactant must be non-toxic and is preferably soluble in the propellant. Typical examples of such agents include fatty acids having 6 to 22 carbon atoms (e.g., caproic, octanoic, lauric, palmitic, stearic, linoleic, linolenic, olesteric and oleic acids) and aliphatic polyhydric acids. An ester or a partial ester of a polyhydric alcohol or a cyclic anhydride thereof. Mixed esters, for example mixed or natural glycerides, may be used. Surfactants may be present in the composition from 0.1% by weight to
It will account for 20% by weight, preferably 0.25-5% by weight. The equilibration of the composition is usually carried out with a propellant. A carrier may be included, as desired, and nasal administration may include lecithin.

In another aspect, the present invention relates to a vaccine comprising an immunologically effective amount of an immunogenic peptide described herein as an active ingredient. Such peptides can be introduced into a host, such as a human, linked to its own carrier, or as a homopolymer or heteropolymer of active peptide units. Such polymers have the advantage of enhanced immunological response and, when different peptides are used for the construction of this polymer, induce antibodies and / or CTLs which react with different antigenic determinants of the virus or tumor cells. It has the advantage of additional capabilities. Useful carriers are well known in the art and include, for example, thyroglobulin, albumin such as human serum albumin, tetanus toxin, polyamino acids such as poly (lysine: glutamic acid), inflenza, hepatitis B virus core protein, hepatitis B virus. And recombinant vaccines. These vaccines may further comprise a physiologically tolerable (acceptable) diluent, such as water, phosphate buffered saline, or saline, and more typically comprise an adjuvant. Adjuvants such as incomplete Freund's adjuvant, aluminum phosphate, aluminum hydroxide or alum are materials well known in the art. In addition, as described above, the CTL response can be achieved by converting the peptide of the present invention to a lipid such as P ₃ CS
It can be primed by bonding to S. Immunization via injection, aerosol, oral, transdermal or other routes with the peptide compositions described herein causes the host immune system to produce large amounts of CTL specific for the desired antigen against this vaccine. And the host is at least partially immunized against subsequent infection or becomes resistant to the development of a chronic infection.

A vaccine composition comprising a peptide of the invention is administered to a patient susceptible or otherwise susceptible to a viral infection or cancer, and induces an immune response to the antigen, thereby Enhance the patient's own immune response ability. Such an amount is defined as an "immunologically effective dose." The exact amount for use will again depend on the patient's state of health and weight, the mode of administration, the type of formulation, etc., but the general range is from about 1.0 μg to about 5,000 μg per 70 kg patient. More usually about 10 μg to about 500 μg mg per 70 kg patient.

In some situations, it may be desirable to combine the peptide vaccines of the invention with a vaccine that induces a neutralizing antibody response against the virus of interest, particularly the viral envelope antigen. Nucleic acids encoding one or more peptides of the invention may be administered to a patient for therapeutic or immunological purposes. Several methods are suitably used for introducing nucleic acids into a patient. For example, nucleic acids can be introduced directly as "naked DNA." This approach is described, for example, in Wolff et al., Science 247: 1465-1468 (1990) and US Pat.
No. 5,580,859 and No. 5,589,466. Nucleic acids may also be administered utilizing ballistic transfer, for example, as described in US Pat. No. 5,204,253. Particles containing only DNA may be administered. On the other hand,
DNA may be attached to particles, for example, gold particles. The nucleic acid may be introduced by being complexed with a cationic compound, for example, a cationic lipid. Lipid-mediated gene transfer methods include, for example, W
WO96 / 18372; WO93 / 24640; Mannino and Gould-Fogerite (19
88) BioTechniques 6 (7): 682-691; Rose U.S. Pat. No. 5,279,833;
WO91 / 06303; and Felgner et al. (1987) Proc. Natl. Acad. Sci. USA 8
4: 7413-7414. The peptides of the invention may also be expressed via an attenuated viral host, such as vaccinia or fowlpox. This approach involves the use of vaccinia virus as a vector to express a nucleotide sequence encoding a peptide of the invention. Upon introduction into an acute or chronic infected or uninfected host, the recombinant vaccinia virus expresses an immunogenic peptide and thus elicits a CTL response in the host. Vaccinia vectors and methods useful for immunization protocols are described, for example, in US Pat. No. 4,722,848, which is incorporated herein by reference. Other vectors are BCG
(Bacille Calmette Guerin). The BCG vector is described by Stover et al. ( Nature 351: 456-460 (herein incorporated by reference).
1991)). A wide variety of other vectors useful for therapeutic administration or immunization of the peptides of the invention, such as the Salmonella typ hi vector, will be apparent to those skilled in the art from the description herein. .

A preferred means of administering a nucleic acid encoding a peptide of the invention utilizes a minigene construct encoding a multiple epitope of the invention. To construct a DNA sequence (minigene) encoding a CTL epitope of choice for expression in human cells, the amino acid sequence of this epitope is reverse transcribed. Use the human codon usage table to guide codon selection for each amino acid. The DNA sequences encoding such epitopes are linked directly to form a continuous polypeptide sequence. Additional elements may be incorporated into this minigene design to optimize expression and / or immunogenicity. Examples of amino acid sequences that are reverse transcribed and can be included in the minigene sequence include: helper T lymphocyte epitopes, leader (signal) sequences, and ER anchoring sequences. Furthermore, the CTL epitope M
HC presentation can be enhanced by including synthetic (eg, polyalanine) or natural flanking sequences next to the CTL epitope.

The minigene sequence is converted to DNA by assembling oligonucleotides encoding the positive and negative strands of the minigene. Utilizing well-known techniques, overlapping oligonucleotides (30-100 bases in length) are synthesized, phosphorylated, purified, and annealed under appropriate conditions. Oligonucleotide ends are T4 DN
Ligation using A ligase. This synthetic minigene encoding a CTL epitope polypeptide can then be cloned into the desired expression vector.

[0073] Standard regulatory sequences well known to those skilled in the art are included in the vector to ensure expression in target cells. Several vector elements are required: a promoter with a downstream cloning site for minigene insertion; a polyadenylation signal for efficient transcription termination; An E. coli origin of replication; Coli selectable marker (eg, ampicillin or kanamycin resistance). Numerous promoters can be used for this purpose, for example the human cytomegalovirus (hCMV) promoter. For other suitable promoter sequences, see US Pat.
See 580,859 and 5,589,466.

[0074] Additional vector modifications may be desired to optimize minigene expression and immunogenicity. In some situations, introns are required for efficient gene expression, and one or more synthetic or natural introns may be incorporated into the transcribed region of the minigene. Incorporation of mRNA stabilizing sequences may also be considered for minigene expression. Immunostimulatory sequences (ISS or CpGs) in the immunogenicity of DNA vaccines
It has recently been suggested that is playing a role. Such a sequence may be included in the vector, outside of the minigene coding sequence, if it has been found to enhance immunogenicity.

In some embodiments, a bicistronic expression vector can be used that allows for the production of a minigene-encoding epitope and a second protein to be included to enhance or reduce immunogenicity. Examples of proteins or polypeptides that can advantageously enhance an immune response when co-expressed include cytokines (eg, IL2, IL
12, GM-CSF), cytokine-inducing molecules (eg, LeLF), or costimulatory molecules. Helper (HTL) epitopes can be linked to an intracellular targeting signal and expressed independently of the CTL epitope. This will allow the induction of HTL epitopes into cell compartments other than CTL epitopes. Where appropriate, this facilitates more efficient entry of HTL epitopes into the MHC class II pathway, and thus enhances CTL induction. Contrary to CTL induction, a specific decrease in the immune response by co-expression of an immunosuppressive molecule (eg, TGF-β) may be advantageous in certain diseases.

Once the expression vector has been selected, the minigene is cloned into the polylinker region downstream of the promoter. This plasmid is transformed into an appropriate E. coli. The E. coli strain is transformed and DNA is prepared using standard techniques. Restriction mapping and DN mapping of the orientation and DNA sequence of the minigene and other elements contained within this vector.
Confirm by A sequence analysis. Bacterial cells on which the proper plasmid has been established can be stored as a master cell bank and a working cell bank.

Therapeutic amounts of plasmid DNA were obtained from E. coli. It is produced by fermentation of E. coli and subsequent purification. An aliquot from the working cell bank is transferred to a fermentation medium (eg, Terrific Broth
) And grown to saturation in a shaker flask or bioreactor according to well known techniques. Plasmid DNA can be purified using standard bioseparation techniques, for example, a solid phase anion exchange resin supplied by Quiagen.
If necessary, supercoiled DNA can be isolated from the open or linear form using gel electrophoresis or other methods.

[0078] Purified plasmid DNA can be prepared for injection using a variety of formulations. The simplest is the reconstitution of lyophilized DNA in sterile phosphate buffered saline (PBS). Various methods have been announced and new technologies have become useful. As mentioned above, nucleic acids are easily formulated with cationic lipids. In addition, glycolipids, fusogenic liposomes, peptides and compounds, collectively referred to as protective, interactive, non-condensable (PINC), are conjugated to purified plasmid DNA to produce variables, such as stability,
It can affect intramuscular dispersibility, or transport to a particular organ or cell type.

Sensitization of target cells may be used for the expression of minigene-encoded CTL epitopes and for functional assays of MHC class I presentation. Plasmid DNA was converted to standard CT
Introduce into a suitable mammalian cell line as a target for the L chromium release assay. The transfection method used will depend on the final formulation. "Nude" DN
Electroporation can be used for A, while cationic lipids are directly
Enables in vitro transfection. Co-transfection of a plasmid expressing green fluorescein protein (GFP) and enrichment of the transfected cells is possible by utilizing fluorescent activated cell sorting (FACS). Such cells are then chromium-51 labeled and used as target cells for an epitope-specific CTL line. Cell lysis detected by 51Cr release suggests a consequence of MHC presentation of the minigene-encoded CTL epitope.

In vivo immunogenicity is a second approach for functional testing of minigene DNA preparations. Transgenic mice expressing appropriate human MHC molecules were
Immunize with the A product. The dose and route of administration are formulation dependent (eg, PBS
DNA inside is 1M, lipid complex DNA is 1P). Twenty-one days after immunization, spleen cells are harvested and restimulated for one week in the presence of the peptide encoding each epitope to be tested. These effector cells (CTL) are assayed for cell lysis of peptide-loaded chromium-51 labeled target cells using standard techniques. Lysis of target cells sensitized by MHC loading with a peptide corresponding to the minigene-encoded epitope demonstrates the function of the DNA vaccine for in vivo induction of CTL.

[0081] Antigenic peptides can also be used to induce CTL ex vivo. The resulting CTLs can be used to treat chronic infections (viral or bacterial) or tumors in patients that will not respond to other conventional forms of treatment or to peptide vaccine treatment approaches. Ex vivo CTL responses to specific pathogens (infectious agents or tumor antigens) are expressed in tissue culture in patient CTL precursor cells (
CTLp) is derived by incubation with the source of antigen presenting cells (APC) and the appropriate immunogenic peptide. CTLp is activated and matures,
Then, after a suitable incubation time to develop into effector CTL (typically 1-4 weeks), the cells are returned to the patient, where they destroy their specific target cells (infected or tumor cells). Will.

These peptides may also find use as diagnostic reagents. For example, the peptides of the present invention can be used to determine the susceptibility of a particular individual to a treatment regimen employing the peptide or related peptides, and thus to revise current treatment protocols, or It may be useful to determine the prognosis of the affected individual. In addition, these peptides can be used to predict individuals who will be at considerable risk for developing chronic infections.

The following examples are illustrative and do not limit the invention. Example 1 Isolation of a class I antigen was performed as described in the aforementioned related application. The naturally processed peptide was isolated and sequenced as described therein. Allele specific motifs and algorithms were also determined and quantitative binding assays were performed.

Utilizing the above identified motifs for various HLA alleles, amino acid sequences from a number of antigens were analyzed for the presence of these motifs. Table 3- ^** provides the results of these searches. The above examples are illustrative of the invention and do not limit the scope of the invention. Other variants of the invention will be apparent to those skilled in the art and are encompassed by the present invention. All publications, patents and patent applications are incorporated herein by reference.

[0085]

[Table 3]

[0086]

[Table 4]

[0087]

[Table 5]

[0088]

[Table 6]

[0089]

[Table 7]

[0090]

[Table 8]

[0091]

[Table 9]

[0092]

[Table 10]

[0093]

[Table 11]

[0094]

[Table 12]

[0095]

[Table 13]

[0096]

[Table 14]

[0097]

[Table 15]

[0098]

[Table 16]

[0099]

[Table 17]

[0100]

[Table 18]

[0101]

[Table 19]

[0102]

[Table 20]

[0103]

[Table 21]

[0104]

[Table 22]

[0105]

[Table 23]

[0106]

[Table 24]

[0107]

[Table 25]

[0108]

[Table 26]

[0109]

[Table 27]

[0110]

[Table 28]

[0111]

[Table 29]

[0112]

[Table 30]

[0113]

[Table 31]

[0114]

[Table 32]

[0115]

[Table 33]

[0116]

[Table 34]

[0117]

[Table 35]

[0118]

[Table 36]

[0119]

[Table 37]

[0120]

[Table 38]

[0121]

[Table 39]

[0122]

[Table 40]

[0123]

[Table 41]

[0124]

[Table 42]

[0125]

[Table 43]

[0126]

[Table 44]

[0127]

[Table 45]

[0128]

[Table 46]

[0129]

[Table 47]

[0130]

[Table 48]

[0131]

[Table 49]

[0132]

[Table 50]

[0133]

[Table 51]

[0134]

[Table 52]

[0135]

[Table 53]

[0136]

[Table 54]

[0137]

[Table 55]

[0138]

[Table 56]

[0139]

[Table 57]

[0140]

[Table 58]

[0141]

[Table 59]

[0142]

[Table 60]

[0143]

[Table 61]

[0144]

[Table 62]

[0145]

[Table 63]

[0146]

[Table 64]

[0147]

[Table 65]

[0148]

[Table 66]

[0149]

[Table 67]

[0150]

[Table 68]

[0151]

[Table 69]

[0152]

[Table 70]

[0153]

[Table 71]

[0154]

[Table 72]

[0155]

[Table 73]

[0156]

[Table 74]

[0157]

[Table 75]

[0158]

[Table 76]

[0159]

[Table 77]

[0160]

[Table 78]

[0161]

[Table 79]

[0162]

[Table 80]

[0163]

[Table 81]

[0164]

[Table 82]

[0165]

[Table 83]

[0166]

[Table 84]

[0167]

[Table 85]

[0168]

[Table 86]

[0169]

[Table 87]

[0170]

[Table 88]

[0171]

[Table 89]

[0172]

[Table 90]

[0173]

[Table 91]

[0174]

[Table 92]

[0175]

[Table 93]

[0176]

[Table 94]

[0177]

[Table 95]

[0178]

[Table 96]

[0179]

[Table 97]

[0180]

[Table 98]

[0181]

[Table 99]

[0182]

[Table 100]

[0183]

[Table 101]

[0184]

[Table 102]

[0185]

[Table 103]

[0186]

[Table 104]

[0187]

[Table 105]

[0188]

[Table 106]

[0189]

[Table 107]

[0190]

[Table 108]

[0191]

[Table 109]

[0192]

[Table 110]

[0193]

[Table 111]

[0194]

[Table 112]

[0195]

[Table 113]

[0196]

[Table 114]

[0197]

[Table 115]

[0198]

[Table 116]

[0199]

[Table 117]

[0200]

[Table 118]

[0201]

[Table 119]

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C07K 7/00 C12Q 1/02 C12Q 1/02 C07K 14/705 // C07K 14/705 C12N 15/00 ZNAA (81) Designated countries EP (AT, BE, CH, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF) , CG, CI, CM, GA, GN, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, C U, CZ, DE, DK, EE, ES, FI, GB, GE, GH, GM, GW, HU, ID, IL, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR , LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, UZ, VN, YU, ZW (72) Inventor Sydney, John United States of America, California 92037, La Jolla, De. Villa La Jo La Drive 8541 (72) Inventor Celis, Esteban United States, California 92130, San Diego, Land Fair Road 13644 (72) Inventor Gray, Hallaward M. United States, California 92037, La Jolla, La Jolla Street 9066 (72) Inventor Southwood, Scott United States, California 92071, Santee, Straymore Drive 10679 F-term (reference) 4B024 AA01 AA12 AA14 CA05 DA02 DA05 DA12 EA04 GA11 HA01 4B063 QA01 QA18 QA19 QQ02 QQ96 QR48 QS22 4C085 AA03 AA08 BA03 BA09 BA69 BA78 BA87 BA89 BB01 BB11 CC07 CC08 4H045 AA10 BA10 BA41 EA31 FA10 FA74

Claims (7)

[Claims] 1. A composition comprising an immunogenic peptide having an HLA binding motif, wherein the immunogenic peptide is a peptide shown in Tables 3 to 14 or Tables 3 to 1.
The composition as described above, which is a peptide comprising conservative substitutions of the residues in the peptide shown in 4. 2. The composition of claim 1, wherein said immunogenic peptide is conjugated to a second oligopeptide. 3. The composition of claim 2, wherein said second oligopeptide is a peptide that elicits a helper T response. 4. A composition comprising a nucleic acid molecule encoding an immunogenic peptide shown in Tables 3-14, or a peptide comprising conservative substitutions of the residues of the peptides shown in Tables 3-14. 5. The composition of claim 4, wherein said nucleic acid further comprises a sequence encoding a second immunogenic peptide. 6. The composition of claim 4, wherein said nucleic acid further comprises a sequence encoding an oligopeptide that elicits a helper T response. 7. A method for inducing cytotoxic T cells, comprising contacting cytotoxic T cells with the peptide of claim 1.